Two approaches have been used in efforts to discover novel chemicals useful in medicine, agriculture, or basic research. In the first approach of rational design, researchers perform structural studies to determine the three-dimensional structure of a target molecule in order to design compounds which are likely to interact with that structure. In the second approach, large libraries of compounds are screened for a desired biological activity. Compounds exhibiting activity in these screening assays become lead chemical compounds. Further study of compounds with structural similarity to the lead compounds can then lead to the discovery of other compounds with optimal activity.
Although traditional screening assays have focused on the screening of naturally occurring compounds, the ability to synthesize large combinatorial libraries of compounds with diverse structures has greatly increased the number of compounds available for screening. In combinatorial chemistry, each reactant from a first group of reactants is reacted with each reactant from a second group of reactants to yield products containing all the combinations possible from the reaction. If desired, all of the products from the first reaction are then reacted with each reactant from a third group of reactants to yield a large array of products. Additional reactions, if desired, can further increase the size of the library of compounds. Where it is desirable to use protection/deprotection protocols to prevent reactive groups from participating in a given reaction step, typically the same protocols are used for each compound in the growing library.
The generation and use of combinatorial chemical libraries for the identification of novel lead compounds or for the optimization of a promising lead candidate has emerged as a promising and potentially powerful method for the acceleration of the drug discovery process. (Terrett, N. K., et al., Tetrahedron 51:8135 (1995); Gallop, M. A., et al., J. Med. Chem. 37:1385 (1994); Janda, K. D., Proc. Natl. Acad. Sci. U.S.A. 91:10779 (1994); Pavia, M. R. et al., Bioorg. Med. Chem. Lett. 3:387 (1993)).
Initial studies focused on the synthesis of peptide or oligonucleotide libraries and related oligomeric structures. (See Gallop, supra, Geysen, H. M., et al., Proc. Natl. Acad. Sci. U.S.A. 81:3998 (1984); Lam, K. S., et al., Nature 354:82 (1991); Houghten, R. A., et al., Nature 354:84 (1991); Salmon, S. E. et al., Proc. Natl. Acad. Sci. U.S.A. 90:11708 (1993); Owens, R. A., et al., Biochem. Biophys. Res. Commun. 181:402 (1991); Bock. L. C., et al., Nature 355:564 (1992); Scott, J. K. and Smith, G. P., Science 249:386 (1990); Cwirla, S. E., et al., Proc. Natl. Acad. Sci. U.S.A. 87:6378 (1990); Devlin, J. J., et al., Science 249:404 (1990); Simon, R. J., et al., Proc. Natl. Acad. Sci. U.S.A. 89:9367 (1992); Zuckermann, R. N., et al., J.Am. Chem. Soc. 114:10646 (1992); Miller, S. M., et al., Bioorg. Med. Chem. Lett. 4:2657 (1994); Zuckerman, R. N., et al, J. Med. Chem. 37:2678 (1994); Terrett, N. K., et al., J. Bioorg. Med. Chem. Lett. 5::917 (1995); Cho, C. Y., et al., Science 261:1303 (1993); Winkler et al, WO93/09668 (PCT/US92/10183)); Ostresh, J. M., et al., Proc. Natl. Acad. Sci. U.S.A. 91:11138 (1994).
Because many ligands for biologically important receptors are non-peptide ligands, and because non-peptide compounds can mimic or block the effects of peptide ligands as well as non-peptide ligands, more recent efforts have been directed at exploiting the greater diversity and range of useful properties embodied in more conventional small molecule libraries. (See. e.g., Simon, R. J., et al., Proc. Natl. Acad. Sci. U.S.A. 89:9367 (1992); Zuckermann, R. N., et al., J.Am. Chem. Soc. 114:10646 (1992); Miller, S. M., et al., Bioorg. Med. Chem. Lett. 4:2657 (1994); Zuckerman, R. N., et al, J. Med. Chem. 37:2678 (1994); Terrett, N. K., et al., J. Bioorg. Med. Chem. Lett. 5::917 (1995); Cho, C. Y., et al., Science 261:1303 (1993); Winkler et al, WO93/09668 (PCT/US92/10183)); Ostresh, J. M., et al., Proc. Natl. Acad. Sci. U.S.A. 91:11138 (1994); Bunin, et al., J. Am. Chem. Soc. 114:10997 (1992); Bunin, et al., Proc. Natl. Acad. Sci. U.S.A. 91:4708 (1994); Virgilio, A. A. and Ellman, J. A., J. Am. Chem. Soc. 116:11580 (1994); Kick, E. K., and Ellman, J. A., J. Med. Chem. 38:1427 (1995); DeWitt, S. H., et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993); Chen, C., et al., J. Am. Chem. Soc. 116:2661 (1994); Beebe, X., et al., J. Am. Chem. Soc. 114:10061 (1992); Moon, H. -S., et al., Tetrahedron Lett. 35:8915 (1994); Kurth, M. J., et al., J. Org. Chem. 59:5862 (1994); Gordon, D. W., and Steele, J., J. Bioorg. Med. Chem. Lett. 5;47 (1995); Patek, M., et al., Tetrahedron Lett. 35:9169 (1994); Patek, M., et al., Tetrahedron Lett. 36:2227 (1995); Campbell, D. A., et al., J. Am. Chem. Soc. 117:5381 (1995); Forman, F. W., and Sucholeiki, I., J. Org. Chem. 60:523 (1995); Rano, T. A, and Chapman, K. T., Tetrahedron Lett. 36:37879 (1995); Dankwardt, S. M., et al., Tetrahedron Lett. 36: 4923 (1995); Deprez, B., et al., J. Am. Chem. Soc. 117:5405 (1995); Ellman, U.S. Pat. No. 5,288,514).
A range of approaches to the synthesis of diverse chemical libraries have been disclosed including several methods utilizing solid supports. In solid support synthesis, a first reactant is linked to a solid support. This linkage may include a spacer linker arm connecting a functional group on the first reactant to a functional group on the solid support. Reaction of the first reactant bound to the solid support with a second reactant produces a desired product which is bound to the solid support, while unreacted second reactant remains unbound in solution. If desired, additional reactants can be added to the product of the first reaction in subsequent reactions.
Solid phase synthesis has been adapted from solid phase synthesis of peptides and oligonucleotides for use in the synthesis of small chemical libraries. Methods of synthesizing diverse chemical libraries on solid supports include split or mixed synthesis (Furka, A., et al., Abst. 14th Intl. Congress Biochem., Prague 5:47 (1988); Furka, A., et al., Int. J. Peptide Protein Res. 37:487 (1991); Houghten, R. A., Proc. Natl. Acad. Sci. U.S.A. 82:5131 (1985)); Erb, E., et al., Proc. Natl. Acad. Sci. U.S.A. 91:11422 (1994)), encoded synthesis (Brenner, S., and Lerner, R. A., Proc. Natl. Acad. Sci. U.S.A. 89:5381 (1992); Nielsen, J., et al., J. Am. Chem. Soc. 115:9812 (1993); Needels, M. C., et al., Proc. Natl. Acad. Sci. U.S.A. 90:10700 (1993); Nikolaiev, V., et al., Peptide Res. 6:161 (1993); Kerr, J. M., et al., J. Am. Chem. Soc. 115:2529 (1993); Ohlmeyer, M. H. J., et al., Proc. Natl. Acad. Sci. U.S.A. 90:10922 (1993); Nestler, et al., J. Org. Chem. 59:4723 (1994); Baldwin, J. J., et al., J. Am. Chem. Soc. 117:5588 (1995)), indexed synthesis (Pirrung, M. C. and Chen, J., J. Am. Chem. Soc. 117:1240 (1995); Smith, P. W., et al., Bioorg. Med. Chem. Lett. 4:2821 (1994)), or parallel and spatially addressed synthesis on pins (Geysen, et al., Proc. Natl. Acad. Sci. U.S.A. 81:3998 (1984); DeWitt, S. H., et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993)), beads (Merrifield, R. B., J. Am. Chem. Soc. 85:2149 (1963)), chips (Fodor, S. P. A., et al., Science 251: 767 (1991)), and other solid supports (Atherton, E. and Sheppard, R. C., Solid Phase Peptide Synthesis: A Practical Approach (IRL Press: Oxford, 1989); Grubler, G., et al., in Peptides: Chemistry, Structure, and Biology (Proceedings of the Thirteenth American Peptide Symposium) (Hodges, R. A. and Smith, J. A., Eds., ESCOM-Leiden, The Netherlands, 1994) at 51; Englebretsen, D. R. and Harding, D. R. K., Int. J. Peptide Protein Res. 40:487 (1992); Frank, R., Bioorg. Med. Chem. Lett. 3:425 (1993); Frank, R. and Doring, R. Tetrahedron 44:031 (1988); Schmidt. M., et al., Bioorg. Med. Chem. Lett. 3:441 (1993); Eichler, J., et al., Peptide Res. 4:296 (1991)).
Some of the features of solid phase synthesis responsible for its widespread use in chemical synthesis are its repetitive coupling reactions as well as ease of product isolation and sample manipulation. Because the growing product is bound to the solid support, unreacted reactants can be easily removed by washing and/or filtration after each reaction in the synthesis of the final product. Furthermore, because of the ease of removal of unreacted reactants, the synthesis and separation of product from unreacted reactants can be automated. In addition, the ability to isolate the resin bound product by simple filtration permits the use of large reagent excesses to obtain high yields which are required for each step of a multistep synthesis.
In part, because of these features of solid phase synthesis, solution phase combinatorial synthesis has not yet gained wide acceptance as an alternative to solid phase synthesis. There have been, however, recent reports of solution phase, single-step amide, ester or carbamate condensations in the preparation of library mixtures. (Pirrung, M. C. and Chen, J. J. Am. Chem. Soc. 117:1240 (1995); Smith, P. W., et al., Bioorg. Med. Chem. Lett. 4:2821 (1994); Peterson, J. B. in Exploiting Molecular Diversity: Small Molecule Libraries for Drug Discovery, La Jolla, Calif., (Jan. 23-25, 1995). For the introduction of soluble polymer supports, see Han, H., et al., Proc. Natl. Acad. Sci. U.S.A. 92:641(1995)). Methods for carrying out liquid phase synthesis of libraries of peptides and oligonucleotides coupled to a soluble oligomeric support have been described. (Bayer, Ernst and Mutter, Manfred, Nature 237:512-513 (1972); Bayer, Ernst, et al., J. Am. Chem. Soc. 96:7333-7336 (1974); Bonora, G. M., et al., Nucleic Acids Res. 18:3155-3159 (1990)). In oligomer-supported liquid phase synthesis the growing product is attached to a large soluble polymeric group. The product from each step of the synthesis can then be separated from unreacted reactants based on the large difference in size between the relatively large polymer-attached product and the unreacted reactants. This permits reactions to take place in homogeneous solutions, as well as eliminating tedious purification steps associated with traditional liquid phase synthesis. oligomer-supported liquid phase synthesis has also been adapted to automatic liquid phase synthesis of peptides. (Bayer, Ernst, et al., Peptides: Chemistry, Structure, Biology, 426-432).
Liquid phase synthesis also has features which make it attractive for use in chemical synthesis. Liquid phase synthesis does not have the restrictions of scale of reaction imposed by high cost and difficulty in handling large amounts of solid support necessary to obtain large quantities of product. Liquid phase synthesis also eliminates the requirement for the presence of functional groups on the first reactant and the solid support for attachment of the reactant to the solid support or soluble oligomer. (Pirrung, M. C., and Chen, J., J. Am. Chem. Soc. 117:1240 (1995); Smith, P. W., et al., Bioorg. Med. Chem. Lett. 4:2821 (1994)). In addition, the use of liquid phase synthesis also avoids the requirement for compatible spacer linkers. Moreover, liquid phase synthesis, unlike solid phase synthesis, does not require limited reaction chemistries to avoid detachment of the growing product from the solid support, or orthogonal attachment and detachment chemistries which often result in the release of spectator functional groups.
Liquid phase synthesis also does not require the use of specialized protocols for monitoring the individual steps of a multistep solid phase synthesis. (Egner, B. J., et al., J. Org. Chem. 60:2652 (1995); Anderson, R. C., et al., J. Org. Chem. 60:2650 (1995); Fitch, W. L., et al., J. Org. Chem. 59:7955 (1994); Look, G. C., et al., J. Org. Chem. 49:7588 (1994); Metzger, J. W., et al., Angew. Chem., Int. Ed. Engl. 32:894 (1993); Youngquist, R. S., at al., Rapid Commun. Mass Spect. 8:77 (1994); Chu, Y. -H., et al., J. Am. Chem. Soc. 117:5419 (1995); Brummel, C. L., et al., Science 264:399 (1994); Stevanovic, S., et al., Bioorg. Med. Chem. Lett. 3:431 (1993)). In solid phase synthesis, immobilized reactants which fail to react cannot be separated from immobilized reaction product intermediates. If the unreacted reactants participate in later reactions, they will give rise to a different undesired product than the intermediates, and the desired product will be released in an impure state. Thus, to be useful, each reaction in a solid phase synthesis must proceed with an unusually high efficiency. Optimization of the reactions to obtain the required reaction efficiencies is both time consuming and challenging. Even a modest level of purity in the final product (85%) pure requires a 92% yield at each step of a two-step reaction sequence, and a 95% yield at each step of a three-step reaction sequence. These high yields are not routinely available and require both an extensive investment in reaction optimization and/or a purification of the released solid phase product at each step. In addition, it may be necessary to use capping reactions at each step of the reaction to prevent the unreacted reactant from participating in subsequent reactions.
Because intermediates are not immobilized in liquid phase synthesis, liquid phase synthesis permits ease of sample manipulation and the purification of intermediates at each step. The non-limiting scale, expanded and nonlimiting repertoire of chemical reactions, direct production of soluble intermediates and final products for assay or for purification, and the lack of required linking, attachment/detachment or capping strategies make solution phase combinatorial synthesis an attractive alternative to solid phase synthesis.
None of the references described herein is admitted to be prior art.